To investigate mechanisms of phagocytic cell activation we developed methods to measure intracellular calcium (fura2 & indo1) and membrane potential independent of mitochondrial potential (oxonol dyes). A pool of intracellular calcium regulates neutrophil function, a calcium signal is sufficient to stimulate superoxide and secretion, and there is selective inhibition of calcium dependent activation by PMA, possibly through protein kinase c mediated phosphorylation. We studied protein kinase c using the activator di-C8 (diacylglycerol analog). di-C8 stimulates superoxide and secretion like PMA but low doses cause transient responses resembling chemoattractant effects. We investigated the activation of mast cells and lymphocytes. Both calcium dependent and calcium independent activation mechanisms were found in mast cells. We found that a calcium dependent membrane potential depolarization was elicited by IgE binding. These changes reflect a large calcium efflux and its role in activation is now being defined using a series of cell lines with specific biochemical and functional defects. The studies with lymphocytes indicate that both T and B cells display a calcium dependent and activated potassium flux which is sensitive to manipulation in cold or minus calcium media. Studies with T cells and lines transvected with the T cell receptor indicate these cells are activated mitogentically and functional display a rise in calcium when the T cell receptor is crosslinked by antibody.